Various measurements employing fluorescent substances are performed in the field of chemistry. Moreover, in fields such as biotechnology, medicine, and medical care, cells etc. are stained by a fluorescent substance and observed under a fluorescence microscope. Fluorescence assays play an important role in these researches.
Further, in the field of medical care, photodynamic diagnosis (hereinafter abbreviated as PDD) in which a compound that reacts to light (a photosensitizer) is administered and light is irradiated in order to specify the target site is recently gaining attention as a diagnostic method for diseased tissues such as cancer, warts, and acnes. Photodynamic diagnosis (PDD) is, for example, a diagnostic method that specifies the presence or absence of illnesses and affected sites by administering a porphyrin- or a chlorin-based photosensitizer to a patient, allowing this to be accumulated at the diseased tissue, irradiating light at a wavelength of around 400 nm to allow emission of fluorescence, and observing this. Photodynamic diagnosis (PDD) has the advantage of placing less burden on patients by virtue of low invasion and less side effects compared to prior diagnostic methods.
In addition, a method is also being developed in which 5-aminolevulinic acids (ALAs) are administered as the photosensitizer employed in this photodynamic diagnosis (PDD) instead of administering a porphyrin-based photosensitizer. 5-aminolevulinic acid (ALA) is a type of amino acid, and is a substance that is the sole source material of porphyrins essential for plant chlorophyll, heme in animal blood, or the like. It is known that in animals, 5-aminolevulinic acid is metabolized to porphobilinogen, hydroxymethylbilane, uroporphyrinogen coproporphyrinogen III, protoporphyrinogen IX, and protoporphyrin IX in that order, protoporphyrin coordinates with an iron ion to become a heme, and heme binds with a globin to become a hemoglobin.
Porphyrins are known to be incorporated by tumor cells and accumulated. On the other hand, ALAs are known to be incorporated by tumor cells and accumulated in protoporphyrin IX state via said metabolic pathway. Regardless of which administration is taken, the fluorescence of porphyrin is measured to specify the tumor site for diagnosis.
However, porphyrins employed as a photosensitizer develop photobleaching (also referred to as fluorescence discoloration, fluorescence fading) upon fluorescence measurement, and a decrease in fluorescence intensity is observed over time. Photobleaching is a chemical reaction seen on rare occasions with excited fluorescent dye molecules. This reaction occurs because the fluorescent substance in an excited state becomes chemically activated and unstable compared to the ground state. As a result of this reaction, the fluorescent molecule ultimately becomes a low fluorescent structure. For example, in the case of protoporphyrin IX (hereinafter abbreviated as PpIX), there was a problem that the time frame for diagnosis by fluorescence will be short since fluorescence decreases very rapidly, e.g. fluorescence intensity is reduced to one tenth or less in 60 seconds. Accordingly, a method for inhibiting this photobleaching has been desired. However, a method for inhibiting photobleaching of porphyrins has not been known well.
In the meantime, FITC is listed as a fluorescent substance well-employed in fields such as chemistry and biotechnology. In regards to decrease in FITC fluorescence, there is an example of investigating the effect of inhibiting fluorescence decrease with three types of compounds, p-paraphenylenediamine (referred to as PPD), n-propylgallate (referred to as NPG), or 1,4-diazabicyclo[2.2.2]octane (referred to as DABCO) (see Non-Patent Literature 1). However, it was unknown whether or not these compounds exert a similar effect on porphyrins, which are different fluorescent substances having totally differing structure.